Neurons must extend long axons over highly specific routes to establish their normal connectivity during development. The growing tip of an axon, the growth cone, navigates accurately using inhomogeneously distributed guidance cues in its outside environment. Some of these cues are attractive for specific growth cones while others are repulsive. Recent work suggests that there are a number of developmental systems in which repulsive cues play an important role in growth cone guidance. Repulsive guidance cues that persist in the mature central nervous system may also prevent the regeneration of damaged axons. We have developed a method for the identification of candidate repulsive guidance cues. A bioassay for inhibitors of growth cone motility is used in tandem with standard biochemical separation techniques to purify inhibitory molecules. Using this approach, we have recently purified, cloned, and sequenced a likely repulsive guidance cue named collapsin. Here we propose to use the same approach to identify a novel cue that repulses the growth cones of central neurons from the axons of peripheral neurons. This cue is a serine protease that can be neutralized by an irreversible active-site inhibitor. Using this inhibitor as a guide, a number of affinity chromatography steps have been identified that enrich the repulsive activity. Once this cue is purified, cloned, and sequenced, we will be able to study its distribution and physiological function in axonal pathfinding during normal development. We hypothesize that it plays an important role in the establishment of separate sensory and central axon tracts in the brain stem.